Hypoxia plays a crucial role in regulating vascularization. A commonly accepted theme is that hypoxia drives the expression of various growth factors (such as VEGF-A) by tumor cells, mesenchymal cells etc., which in turn act on blood vessels. In vivo, it is not known if the endothelium per se is directly regulated by hypoxia as well, although in vitro cultured endothelial cells can respond to hypoxia. We have recently discovered that hypoxia-inducible factor 2alpha, whose expression is largely limited to the endothelium during embryogenesis, is important for developmental vascular remodeling. Accordingly, we hypothesize that hypoxia may directly modulate the function of the endothelium. We will test this novel hypothesis in three specific aims. In Aim 1, we will substantiate our observation by confirming that the role of HIF-2alpha indeed resides within the endothelium. We will also investigate if there is a correlation between the expression and functional requirement of HIF-2alpha and the hypoxic status of different subsets of blood vessels. In Aim 2, we will address if the role of HIF-2alpha in regulating vascular development is indeed dependent on its ability to mediate hypoxia response. We will mutate the proline residue in the oxygen dependent degradation domain, and a cysteine residue in the C-terminal activation domain important for its regulation with p300/CBP, an important mediator of the hypoxia pathway. We will test the effect of such mutations on the expression and function of NIF-2alpha during vascular remodeling. In Aim 3, we will characterize the nature of cellular and molecular abnormalities caused by HIF-2alpha deficiency. Specifically, we will determine if HIF-2alpha -/- endothelial cells are abnormal in one or more of endothelial properties, including migration, attachment and tube formation in vitro. At the molecular level we will take a novel proteomic approach to screen for genes downstream to HIF-2alpha and confirm their functions by RNA interference in in vitro assays such as tube formation. Together, these experiments may allow us to uncover a novel mechanism in vascular development.